Controlling CAR-T cell activity and specificity with synthetic SparX adapters.

While conventional chimeric antigen-receptor (CAR)-T therapies have shown remarkable clinical activity in some settings, they can induce severe toxicities and are rarely curative. To address these challenges, we developed a controllable cell therapy where synthetic D-domain-containing proteins (soluble protein antigen-receptor X-linker [SparX]) bind one or more tumor antigens and mark those cells for elimination by genetically modified T cells (antigen-receptor complex [ARC]-T). The chimeric antigen receptor was engineered with a D-domain that specifically binds to the SparX protein via a unique TAG, derived from human alpha-fetoprotein. The interaction is mediated through an epitope on the TAG that is occluded in the native alpha-fetoprotein molecule. In vitro and in vivo data demonstrate that the activation and cytolytic activity of ARC-T cells is dependent on the dose of SparX protein and only occurs when ARC-T cells are engaged with SparX proteins bound to antigen-positive cells. ARC-T cell specificity was also redirected in vivo by changing SparX proteins that recognized different tumor antigens to combat inherent or acquired tumor heterogeneity. The ARC-SparX platform is designed to expand patient and physician access to cell therapy by controlling potential toxicities through SparX dosing regimens and enhancing tumor elimination through sequential or simultaneous administration of SparX proteins engineered to bind different tumor antigens.

MEDI3039, a novel highly potent tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptor 2 agonist, causes regression of orthotopic tumors and inhibits outgrowth of metastatic triple-negative breast cancer.

BACKGROUND: TNF-related apoptosis-inducing ligand (TRAIL) receptor agonists are attractive anti-tumor agents because of their capability to induce apoptosis in cancer cells by activating death receptors (DR) 4 and 5 with little toxicity against normal cells. Despite an attractive mechanism of action, previous clinical efforts to use TRAIL receptor agonists have been unsuccessful. In this study, we examined MEDI3039, a highly potent multivalent DR5 agonist, in breast cancer cell lines and in vivo models. METHODS: As in vitro model systems, we used 19 breast cancer cell lines that are categorized into four subtypes: ER+, HER2 amplified, basal A (triple-negative breast cancer) TNBC, and basal B TNBC. Cell viability was analyzed by MTS and RealTime live/dead assays. As in vivo model systems, MDA-MB231T orthotopic primary tumor growth in the mammary fat pad (MFP) and two experimental lung metastasis models were used. The effect of MEDI3039 on MFP tumors was assessed with immunohistochemical analysis. Lung metastases were analyzed with Bouin's and H&E staining. RESULTS: MEDI3039 killed multiple breast cancer cell lines, but the sensitivity varied among different subtypes. Sensitivity was basal B TNBC >> basal A TNBC > HER2 amplified > ER+ (average IC50 = 1.4, 203, 314, 403 pM, respectively). While the pattern of relative sensitivity was similar to GST-TRAIL in most cell lines, MEDI3039 was at least two orders of magnitude more potent compared with GST-TRAIL. In the MFP model, weekly treatment with 0.1 or 0.3 mg/kg MEDI3039 for 5 weeks inhibited tumor growth by 99.05% or 100% (median), respectively, compared with the control group, and extended animal survival (p = 0.08 or p = 0.0032 at 0.1 or 0.3 mg/kg, respectively). MEDI3039-induced caspase activation was confirmed in tumors grown in MFP (p < 0.05). In an experimental pulmonary metastasis model, MEDI3039 significantly suppressed outgrowth of surface (p < 0.0001) and microscopic metastases (p < 0.05). In an established lung metastasis model, MEDI3039 significantly inhibited growth of metastases (p < 0.01 in surface [> 4 mm], p < 0.01 in tumor percentage) and extended animal survival (p < 0.0001). CONCLUSION: MEDI3039 is a potent DR5 agonist in breast cancer cells in vitro and in vivo and has potential as a cancer drug in breast cancer patients, especially those with basal B TNBC.

Genomic Landscape Survey Identifies SRSF1 as a Key Oncodriver in Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is an aggressive disease with poor survival. A few sequencing studies performed on limited number of samples have revealed potential disease-driving genes in SCLC, however, much still remains unknown, particularly in the Asian patient population. Here we conducted whole exome sequencing (WES) and transcriptomic sequencing of primary tumors from 99 Chinese SCLC patients. Dysregulation of tumor suppressor genes TP53 and RB1 was observed in 82% and 62% of SCLC patients, respectively, and more than half of the SCLC patients (62%) harbored TP53 and RB1 mutation and/or copy number loss. Additionally, Serine/Arginine Splicing Factor 1 (SRSF1) DNA copy number gain and mRNA over-expression was strongly associated with poor survival using both discovery and validation patient cohorts. Functional studies in vitro and in vivo demonstrate that SRSF1 is important for tumorigenicity of SCLC and may play a key role in DNA repair and chemo-sensitivity. These results strongly support SRSF1 as a prognostic biomarker in SCLC and provide a rationale for personalized therapy in SCLC.

Design and Preclinical Evaluation of a Novel B7-H4-Directed Antibody-Drug Conjugate, AZD8205, Alone and in Combination with the PARP1-Selective Inhibitor AZD5305.

PURPOSE: We evaluated the activity of AZD8205, a B7-H4-directed antibody-drug conjugate (ADC) bearing a novel topoisomerase I inhibitor (TOP1i) payload, alone and in combination with the PARP1-selective inhibitor AZD5305, in preclinical models. EXPERIMENTAL DESIGN: IHC and deep-learning-based image analysis algorithms were used to assess prevalence and intratumoral heterogeneity of B7-H4 expression in human tumors. Several TOP1i-ADCs, prepared with Val-Ala or Gly-Gly-Phe-Gly peptide linkers, with or without a PEG8 spacer, were compared in biophysical, in vivo efficacy, and rat toxicology studies. AZD8205 mechanism of action and efficacy studies were conducted in human cancer cell line and patient-derived xenograft (PDX) models. RESULTS: Evaluation of IHC-staining density on a per-cell basis revealed a range of heterogeneous B7-H4 expression across patient tumors. This informed selection of bystander-capable Val-Ala-PEG8-TOP1i payload AZ14170133 and development of AZD8205, which demonstrated improved stability, efficacy, and safety compared with other linker-payload ADCs. In a study of 26 PDX tumors, single administration of 3.5 mg/kg AZD8205 provided a 69% overall response rate, according to modified RECIST criteria, which correlated with homologous recombination repair (HRR) deficiency (HRD) and elevated levels of B7-H4 in HRR-proficient models. Addition of AZD5305 sensitized very low B7-H4-expressing tumors to AZD8205 treatment, independent of HRD status and in models representing clinically relevant mechanisms of PARPi resistance. CONCLUSIONS: These data provide evidence for the potential utility of AZD8205 for treatment of B7-H4-expressing tumors and support the rationale for an ongoing phase 1 clinical study (NCT05123482). See related commentary by Pommier and Thomas, p. 991.

Preclinical Efficacy of BCMA-Directed CAR T Cells Incorporating a Novel D Domain Antigen Recognition Domain.

Chimeric antigen receptor (CAR) T-cell therapies directed against B-cell maturation antigen (BCMA) have shown compelling clinical activity and manageable safety in subjects with relapsed and refractory multiple myeloma (RRMM). Prior reported CAR T cells have mostly used antibody fragments such as humanized or murine single-chain variable fragments or camelid heavy-chain antibody fragments as the antigen recognition motif. Herein, we describe the generation and preclinical evaluation of ddBCMA CAR, which uses a novel BCMA binding domain discovered from our D domain phage display libraries and incorporates a 4-1BB costimulatory motif and CD3-zeta T-cell activation domain. Preclinical in vitro studies of ddBCMA CAR T cells cocultured with BCMA-positive cell lines showed highly potent, dose-dependent measures of cytotoxicity, cytokine production, T-cell degranulation, and T-cell proliferation. In each assay, ddBCMA CAR performed as well as the BCMA-directed scFv-based C11D5.3 CAR. Furthermore, ddBCMA CAR T cells demonstrated in vivo tumor suppression in three disseminated BCMA-expressing tumor models in NSG-immunocompromised mice. On the basis of these promising preclinical data, CART-ddBCMA is being studied in a first-in-human phase I clinical study to assess the safety, pharmacokinetics, immunogenicity, efficacy, and duration of effect for patients with RRMM (NCT04155749).

Functional Genomic Identification of Predictors of Sensitivity and Mechanisms of Resistance to Multivalent Second-Generation TRAIL-R2 Agonists.

Multivalent second-generation TRAIL-R2 agonists are currently in late preclinical development and early clinical trials. Herein, we use a representative second-generation agent, MEDI3039, to address two major clinical challenges facing these agents: lack of predictive biomarkers to enable patient selection and emergence of resistance. Genome-wide CRISPR knockout screens were notable for the lack of resistance mechanisms beyond the canonical TRAIL-R2 pathway (caspase-8, FADD, BID) as well as p53 and BAX in TP53 wild-type models, whereas a CRISPR activatory screen identified cell death inhibitors MCL-1 and BCL-XL as mechanisms to suppress MEDI3039-induced cell death. High-throughput drug screening failed to identify genomic alterations associated with response to MEDI3039; however, transcriptomics analysis revealed striking association between MEDI3039 sensitivity and expression of core components of the extrinsic apoptotic pathway, most notably its main apoptotic effector caspase-8 in solid tumor cell lines. Further analyses of colorectal cell lines and patient-derived xenografts identified caspase-8 expression ratio to its endogenous regulator FLIP(L) as predictive of sensitivity to MEDI3039 in several major solid tumor types and a further subset indicated by caspase-8:MCL-1 ratio. Subsequent MEDI3039 combination screening of TRAIL-R2, caspase-8, FADD, and BID knockout models with 60 compounds with varying mechanisms of action identified two inhibitor of apoptosis proteins (IAP) that exhibited strong synergy with MEDI3039 that could reverse resistance only in BID-deleted models. In summary, we identify the ratios of caspase-8:FLIP(L) and caspase-8:MCL-1 as potential predictive biomarkers for second-generation TRAIL-R2 agonists and loss of key effectors such as FADD and caspase-8 as likely drivers of clinical resistance in solid tumors.

Resistance to Pyrrolobenzodiazepine Dimers Is Associated with SLFN11 Downregulation and Can Be Reversed through Inhibition of ATR.

Resistance to antibody-drug conjugates (ADCs) has been observed in both preclinical models and clinical studies. However, mechanisms of resistance to pyrrolobenzodiazepine (PBD)-conjugated ADCs have not been well characterized and thus, this study was designed to investigate development of resistance to PBD dimer warheads and PBD-conjugated ADCs. We established a PBD-resistant cell line, 361-PBDr, by treating human breast cancer MDA-MB-361 cells with gradually increasing concentrations of SG3199, the PBD dimer released from the PBD drug-linker tesirine. 361-PBDr cells were over 20-fold less sensitive to SG3199 compared with parental cells and were cross-resistant to other PBD warhead and ADCs conjugated with PBDs. Proteomic profiling revealed that downregulation of Schlafen family member 11 (SLFN11), a putative DNA/RNA helicase, sensitizing cancer cells to DNA-damaging agents, was associated with PBD resistance. Confirmatory studies demonstrated that siRNA knockdown of SLFN11 in multiple tumor cell lines conferred reduced sensitivity to SG3199 and PBD-conjugated ADCs. Treatment with EPZ011989, an EZH2 inhibitor, derepressed SLFN11 expression in 361-PBDr and other SLFN11-deficient tumor cells, and increased sensitivity to PBD and PBD-conjugated ADCs, indicating that the suppression of SLFN11 expression is associated with histone methylation as reported. Moreover, we demonstrated that combining an ataxia telangiectasia and Rad3-related protein (ATR) inhibitor, AZD6738, with SG3199 or PBD-based ADCs led to synergistic cytotoxicity in either resistant 361-PBDr cells or cells that SLFN11 was knocked down via siRNA. Collectively, these data provide insights into potential development of resistance to PBDs and PBD-conjugated ADCs, and more importantly, inform strategy development to overcome such resistance.

Antibody-Drug Conjugates: Future Directions in Clinical and Translational Strategies to Improve the Therapeutic Index.

Since the first approval of gemtuzumab ozogamicin (Mylotarg; Pfizer; CD33 targeted), two additional antibody-drug conjugates (ADC), brentuximab vedotin (Adcetris; Seattle Genetics, Inc.; CD30 targeted) and inotuzumab ozogamicin (Besponsa; Pfizer; CD22 targeted), have been approved for hematologic cancers and 1 ADC, trastuzumab emtansine (Kadcyla; Genentech; HER2 targeted), has been approved to treat breast cancer. Despite a clear clinical benefit being demonstrated for all 4 approved ADCs, the toxicity profiles are comparable with those of standard-of-care chemotherapeutics, with dose-limiting toxicities associated with the mechanism of activity of the cytotoxic warhead. However, the enthusiasm to develop ADCs has not been dampened; approximately 80 ADCs are in clinical development in nearly 600 clinical trials, and 2 to 3 novel ADCs are likely to be approved within the next few years. While the promise of a more targeted chemotherapy with less toxicity has not yet been realized with ADCs, improvements in technology combined with a wealth of clinical data are helping to shape the future development of ADCs. In this review, we discuss the clinical and translational strategies associated with improving the therapeutic index for ADCs.

Improved Therapeutic Window in BRCA-mutant Tumors with Antibody-linked Pyrrolobenzodiazepine Dimers with and without PARP Inhibition.

Pyrrolobenzodiazepine dimers (PBD) form cross-links within the minor groove of DNA causing double-strand breaks (DSB). DNA repair genes such as BRCA1 and BRCA2 play important roles in homologous recombination repair of DSB. We hypothesized that PBD-based antibody-drug conjugates (ADC) will have enhanced killing of cells in which homologous recombination processes are defective by inactivation of BRCA1 or BRCA2 genes. To support this hypothesis, we found 5T4-PBD, a PBD-dimer conjugated to anti-5T4 antibody, elicited more potent antitumor activity in tumor xenografts that carry defects in DNA repair due to BRCA mutations compared with BRCA wild-type xenografts. To delineate the role of BRCA1/2 mutations in determining sensitivity to PBD, we used siRNA knockdown and isogenic BRCA1/2 knockout models to demonstrate that BRCA deficiency markedly increased cell sensitivity to PBD-based ADCs. To understand the translational potential of treating patients with BRCA deficiency using PBD-based ADCs, we conducted a "mouse clinical trial" on 23 patient-derived xenograft (PDX) models bearing mutations in BRCA1 or BRCA2 Of these PDX models, 61% to 74% had tumor stasis or regression when treated with a single dose of 0.3 mg/kg or three fractionated doses of 0.1 mg/kg of a PBD-based ADC. Furthermore, a suboptimal dose of PBD-based ADC in combination with olaparib resulted in significantly improved antitumor effects, was not associated with myelotoxicity, and was well tolerated. In conclusion, PBD-based ADC alone or in combination with a PARP inhibitor may have improved therapeutic window in patients with cancer carrying BRCA mutations.

SLC46A3 as a Potential Predictive Biomarker for Antibody-Drug Conjugates Bearing Noncleavable Linked Maytansinoid and Pyrrolobenzodiazepine Warheads.

PURPOSE: Antibody-drug conjugates (ADC) utilizing noncleavable linker drugs have been approved for clinical use, and several are in development targeting solid and hematologic malignancies including multiple myeloma. Currently, there are no reliable biomarkers of activity for these ADCs other than presence of the targeted antigen. We observed that certain cell lines are innately resistant to such ADCs, and sought to uncover the underlying mechanism of resistance. EXPERIMENTAL DESIGN: The expression of 43 lysosomal membrane target genes was evaluated in cell lines resistant to ADCs bearing the noncleavable linker, pyrrolobenzodiazepine payload SG3376, in vitro. The functional relevance of SLC46A3, a lysosomal transporter of noncleavable ADC catabolites whose expression uniquely correlated with SG3376 resistance, was assessed using EPHA2-, HER2-, and BCMA-targeted ADCs and isogenic cells overexpressing or genetically inactivated for SLC46A3. SLC46A3 expression was also examined in patient-derived xenograft and in vitro models of acquired T-DM1 resistance and multiple myeloma bone marrow samples by RT-PCR. RESULTS: Loss of SLC46A3 expression was found to be a mechanism of innate and acquired resistance to ADCs bearing DM1 and SG3376. Sensitivity was restored in refractory lines upon introduction of SLC46A3, suggesting that expression of SLC46A3 may be more predictive of activity than target antigen levels alone. Interrogation of primary multiple myeloma samples indicated a range of SLC46A3 expression, including samples with undetectable levels like multiple myeloma cell lines resistant to BCMA-targeting DM1 and SG3376 ADCs. CONCLUSIONS: Our findings support SLC46A3 as a potential patient selection biomarker with immediate relevance to clinical trials involving these ADCs.

Preclinical evaluation of a GFRA1 targeted antibody-drug conjugate in breast cancer.

Despite recent advances in treatment, breast cancer remains the second-most common cause of cancer death among American women. A greater understanding of the molecular characteristics of breast tumors could ultimately lead to improved tumor-targeted treatment options, particularly for subsets of breast cancer patients with unmet needs. Using an unbiased genomics approach to uncover membrane-localized tumor-associated antigens (TAAs), we have identified glial cell line derived neurotrophic factor (GDNF) family receptor α 1 (GFRA1) as a breast cancer TAA. Immunohistochemistry (IHC) revealed that GFRA1 displays a limited normal tissue expression profile coupled with overexpression in specific breast cancer subsets. The cell surface localization as determined by fluorescence-activated cell sorting (FACS) and the rapid internalization kinetics of GFRA1 makes it an ideal target for therapeutic exploitation as an antibody-drug conjugate (ADC). Here, we describe the development of a pyrrolobenzodiazepine (PBD)-armed, GFRA1-targeted ADC that demonstrates cytotoxicity in GFRA1-positive cell lines and patient-derived xenograft (PDX) models. The safety profile of the rat cross-reactive GFRA1-PBD was assessed in a rat toxicology study to find transient cellularity reductions in the bone marrow and peripheral blood, consistent with known off-target effects of PBD ADC's. These studies reveal no evidence of on-target toxicity and support further evaluation of GFRA1-PBD in GFRA1-positive tumors.

Loss of functional BAP1 augments sensitivity to TRAIL in cancer cells.

Malignant mesothelioma (MM) is poorly responsive to systemic cytotoxic chemotherapy and invariably fatal. Here we describe a screen of 94 drugs in 15 exome-sequenced MM lines and the discovery of a subset defined by loss of function of the nuclear deubiquitinase BRCA associated protein-1 (BAP1) that demonstrate heightened sensitivity to TRAIL (tumour necrosis factor-related apoptosis-inducing ligand). This association is observed across human early passage MM cultures, mouse xenografts and human tumour explants. We demonstrate that BAP1 deubiquitinase activity and its association with ASXL1 to form the Polycomb repressive deubiquitinase complex (PR-DUB) impacts TRAIL sensitivity implicating transcriptional modulation as an underlying mechanism. Death receptor agonists are well-tolerated anti-cancer agents demonstrating limited therapeutic benefit in trials without a targeting biomarker. We identify BAP1 loss-of-function mutations, which are frequent in MM, as a potential genomic stratification tool for TRAIL sensitivity with immediate and actionable therapeutic implications.

Antitumor Activity of MEDI3726 (ADCT-401), a Pyrrolobenzodiazepine Antibody-Drug Conjugate Targeting PSMA, in Preclinical Models of Prostate Cancer.

Prostate-specific membrane antigen (PSMA) is a membrane-bound glutamate carboxypeptidase that is highly expressed in nearly all prostate cancers with the highest expression in metastatic castration-resistant prostate cancer (mCRPC). The prevalence of increased surface expression and constitutive internalization of PSMA make it an attractive target for an antibody-drug conjugate (ADC) approach to treating patients with mCRPC. MEDI3726 (previously known as ADCT-401) is an ADC consisting of an engineered version of the anti-PSMA antibody J591 site specifically conjugated to the pyrrolobenzodiazepine (PBD) dimer tesirine. MEDI3726 specifically binds the extracellular domain of PSMA and, once internalized, releases the PBD dimer to crosslink DNA and trigger cell death. In vitro, MEDI3726 demonstrated potent and specific cytotoxicity in a panel of PSMA-positive prostate cancer cell lines, consistent with internalization and DNA interstrand crosslinking. In vivo, MEDI3726 showed robust antitumor activity against the LNCaP and the castration-resistant CWR22Rv1 prostate cancer cell line xenografts. MEDI3726 also demonstrated durable antitumor activity in the PSMA-positive human prostate cancer patient-derived xenograft (PDX) LuCaP models. This activity correlated with increased phosphorylated Histone H2AX in tumor xenografts treated with MEDI3726. MEDI3726 is being evaluated in a phase I clinical trial as a treatment for patients with metastatic castrate-resistant prostate cancer (NCT02991911). Mol Cancer Ther; 17(10); 2176-86. ©2018 AACR.

Direct targeting of alphavbeta3 integrin on tumor cells with a monoclonal antibody, Abegrin.

The humanized monoclonal antibody Abegrin, currently in phase II trials for treatment of solid tumors, specifically recognizes the integrin alphavbeta3. Due to its high expression on mature osteoclasts, angiogenic endothelial cells, and tumor cells, integrin alphavbeta3 functions in several pathologic processes important to tumor growth and metastasis. Targeting of this integrin with Abegrin results in antitumor, antiangiogenic, and antiosteolytic activities. Here, we exploit the species specificity of Abegrin to evaluate the effects of direct targeting of tumor cells (independent of targeting of endothelia or osteoclasts). Flow cytometry analysis of human tumor cell lines shows high levels of alphavbeta3 on many solid tumors, including cancers of the prostate, skin, ovary, kidney, lung, and breast. We also show that tumor growth of alphavbeta3-expressing tumor cells is inhibited by Abegrin in a dose-dependent manner. We present a novel finding that high-dose administration can actively impair the antitumor activity of Abegrin. We also provide evidence that antibody-dependent cellular cytotoxicity contributes to in vitro and in vivo antitumor activity. Finally, it was observed that peak biological activity of Abegrin arises at serum levels that are consistent with those achieved in clinical trials. These results support a concept that Abegrin can be used to achieve selective targeting of the many tumor cells that express alphavbeta3 integrin. In combination with the well-established concept that alphavbeta3 plays a key role in cancer-associated angiogenesis and osteolytic activities, this triad of activity could provide new opportunities for therapeutic targeting of cancer.

Optoacoustic Detection of Early Therapy-Induced Tumor Cell Death Using a Targeted Imaging Agent.

Purpose: The development of new treatments and their deployment in the clinic may be assisted by imaging methods that allow an early assessment of treatment response in individual patients. The C2A domain of Synaptotagmin-I (C2Am), which binds to the phosphatidylserine (PS) exposed by apoptotic and necrotic cells, has been developed as an imaging probe for detecting cell death. Multispectral optoacoustic tomography (MSOT) is a real-time and clinically applicable imaging modality that was used here with a near infrared (NIR) fluorophore-labeled C2Am to image tumor cell death in mice treated with a TNF-related apoptosis-inducing ligand receptor 2 (TRAILR2) agonist and with 5-fluorouracil (5-FU).Experimental Design: C2Am was labeled with a NIR fluorophore and injected intravenously into mice bearing human colorectal TRAIL-sensitive Colo205 and TRAIL-resistant HT-29 xenografts that had been treated with a potent agonist of TRAILR2 and in Colo205 tumors treated with 5-FU.Results: Three-dimensional (3D) MSOT images of probe distribution showed development of tumor contrast within 3 hours of probe administration and a signal-to-background ratio in regions containing dead cells of >10 after 24 hours. A site-directed mutant of C2Am that is inactive in PS binding showed negligible binding. Tumor retention of the active probe was strongly correlated (R2 = 0.97, P value < 0.01) with a marker of apoptotic cell death measured in histologic sections obtained post mortem.Conclusions: The rapid development of relatively high levels of contrast suggests that NIR fluorophore-labeled C2Am could be a useful optoacoustic imaging probe for detecting early therapy-induced tumor cell death in the clinic. Clin Cancer Res; 23(22); 6893-903. ©2017 AACR.

5T4-Targeted Therapy Ablates Cancer Stem Cells and Prevents Recurrence of Head and Neck Squamous Cell Carcinoma.

Purpose: Locoregional recurrence is a frequent treatment outcome for patients with advanced head and neck squamous cell carcinoma (HNSCC). Emerging evidence suggests that tumor recurrence is mediated by a small subpopulation of uniquely tumorigenic cells, that is, cancer stem cells (CSC), that are resistant to conventional chemotherapy, endowed with self-renewal and multipotency.Experimental Design: Here, we evaluated the efficacy of MEDI0641, a novel antibody-drug conjugate targeted to 5T4 and carrying a DNA-damaging "payload" (pyrrolobenzodiazepine) in preclinical models of HNSCC.Results: Analysis of a tissue microarray containing 77 HNSCC with follow-up of up to 12 years revealed that patients with 5T4high tumors displayed lower overall survival than those with 5T4low tumors (P = 0.038). 5T4 is more highly expressed in head and neck CSC (ALDHhighCD44high) than in control cells (non-CSC). Treatment with MEDI0641 caused a significant reduction in the CSC fraction in HNSCC cells (UM-SCC-11B, UM-SCC-22B) in vitro Notably, a single intravenous dose of 1 mg/kg MEDI0641 caused long-lasting tumor regression in three patient-derived xenograft (PDX) models of HNSCC. MEDI0641 ablated CSC in the PDX-SCC-M0 model, reduced it by five-fold in the PDX-SCC-M1, and two-fold in the PDX-SCC-M11 model. Importantly, mice (n = 12) treated with neoadjuvant, single administration of MEDI0641 prior to surgical tumor removal showed no recurrence for more than 200 days, whereas the control group had 7 recurrences (in 12 mice; P = 0.0047).Conclusions: Collectively, these findings demonstrate that an anti-5T4 antibody-drug conjugate reduces the fraction of CSCs and prevents local recurrence and suggest a novel therapeutic approach for patients with HNSCC. Clin Cancer Res; 23(10); 2516-27. ©2016 AACR.

Preclinical Evaluation of MEDI0641, a Pyrrolobenzodiazepine-Conjugated Antibody-Drug Conjugate Targeting 5T4.

Antibody-drug conjugates (ADC) are used to selectively deliver cytotoxic agents to tumors and have the potential for increased clinical benefit to cancer patients. 5T4 is an oncofetal antigen overexpressed on the cell surface in many carcinomas on both bulk tumor cells as well as cancer stem cells (CSC), has very limited normal tissue expression, and can internalize when bound by an antibody. An anti-5T4 antibody was identified and optimized for efficient binding and internalization in a target-specific manner, and engineered cysteines were incorporated into the molecule for site-specific conjugation. ADCs targeting 5T4 were constructed by site-specifically conjugating the antibody with payloads that possess different mechanisms of action, either a DNA cross-linking pyrrolobenzodiazepine (PBD) dimer or a microtubule-destabilizing tubulysin, so that each ADC had a drug:antibody ratio of 2. The resulting ADCs demonstrated significant target-dependent activity in vitro and in vivo; however, the ADC conjugated with a PBD payload (5T4-PBD) elicited more durable antitumor responses in vivo than the tubulysin conjugate in xenograft models. Likewise, the 5T4-PBD more potently inhibited the growth of 5T4-positive CSCs in vivo, which likely contributed to its superior antitumor activity. Given that the 5T4-PBD possessed both potent antitumor activity as well as anti-CSC activity, and thus could potentially target bulk tumor cells and CSCs in target-positive indications, it was further evaluated in non-GLP rat toxicology studies that demonstrated excellent in vivo stability with an acceptable safety profile. Taken together, these preclinical data support further development of 5T4-PBD, also known as MEDI0641, against 5T4+ cancer indications. Mol Cancer Ther; 16(8); 1576-87. ©2017 AACR.

Differential EphA2 epitope display on normal versus malignant cells.

The EphA2 receptor tyrosine kinase is overexpressed in many different types of human cancers where it functions as a powerful oncoprotein. Dramatic changes in the subcellular localization and function of EphA2 have also been linked with cancer, and in particular, unstable cancer cell-cell contacts prevent EphA2 from stably binding its ligand on the surface of adjoining cells. This change is important in light of evidence that ligand binding causes EphA2 to transmit signals that negatively regulate tumor cell growth and invasiveness and also induce EphA2 degradation. On the basis of these properties, we have begun to target EphA2 on tumor cells using agonistic antibodies, which mimic the consequences of ligand binding. In our present study, we show that a subset of agonistic EphA2 antibodies selectively bind epitopes on malignant cells, which are not available on nontransformed epithelial cells. We also show that such epitopes arise from differential cell-cell adhesions and that the stable intercellular junctions of nontransformed epithelial cells occlude the binding site for ligand, as well as this subset of EphA2 antibodies. Finally, we demonstrate that antibody targeting of EphA2 decreases tumor cell growth as measured using xenograft tumor models and found that the mechanism of antibody action relates to EphA2 protein degradation in vivo. Taken together, these results suggest new opportunities for therapeutic targeting of the large number of different cancers that express EphA2 in a manner that could minimize potential toxicities to normal cells.

Patient-derived xenograft (PDX) tumors increase growth rate with time.

Patient-derived xenograft (PDX) models are frequently used for translational cancer research, and are assumed to behave consistently as the tumor ages. However, growth rate constancy as a function of time is unclear. Notably, variable PDX growth rates over time might have implications for the interpretation of translational studies. We characterized four PDX models through several in vivo passages from primary human head and neck squamous cell carcinoma and salivary gland adenoid cystic carcinoma. We developed a mathematical approach to merge growth data from different passages into a single measure of relative tumor volume normalized to study initiation size. We analyzed log-relative tumor volume increase with linear mixed effect models. Two oral pathologists analyzed the PDX tissues to determine if histopathological feature changes occurred over in vivo passages. Tumor growth rate increased over time. This was determined by repeated measures linear regression statistical analysis in four different PDX models. A quadratic statistical model for the temporal effect predicted the log-relative tumor volume significantly better than a linear time effect model. We found a significant correlation between passage number and histopathological features of higher tumor grade. Our mathematical treatment of PDX data allows statistical analysis of tumor growth data over long periods of time, including over multiple passages. Non-linear tumor growth in our regression models revealed the exponential growth rate increased over time. The dynamic tumor growth rates correlated with quantifiable histopathological changes that related to passage number in multiple types of cancer.

A Potent HER3 Monoclonal Antibody That Blocks Both Ligand-Dependent and -Independent Activities: Differential Impacts of PTEN Status on Tumor Response.

HER3/ERBB3 is a kinase-deficient member of the EGFR family receptor tyrosine kinases (RTK) that is broadly expressed and activated in human cancers. HER3 is a compelling cancer target due to its important role in activation of the oncogenic PI3K/AKT pathway. It has also been demonstrated to confer tumor resistance to a variety of cancer therapies, especially targeted drugs against EGFR and HER2. HER3 can be activated by its ligand (heregulin/HRG), which induces HER3 heterodimerization with EGFR, HER2, or other RTKs. Alternatively, HER3 can be activated in a ligand-independent manner through heterodimerization with HER2 in HER2-amplified cells. We developed a fully human mAb against HER3 (KTN3379) that efficiently suppressed HER3 activity in both ligand-dependent and independent settings. Correspondingly, KTN3379 inhibited tumor growth in divergent tumor models driven by either ligand-dependent or independent mechanisms in vitro and in vivo Most intriguingly, while investigating the mechanistic underpinnings of tumor response to KTN3379, we discovered an interesting dichotomy in that PTEN loss, a frequently occurring oncogenic lesion in a broad range of cancer types, substantially blunted the tumor response in HER2-amplified cancer, but not in the ligand-driven cancer. To our knowledge, this represents the first study ascertaining the impact of PTEN loss on the antitumor efficacy of a HER3 mAb. KTN3379 is currently undergoing a phase Ib clinical trial in patients with advanced solid tumors. Our current study may help us optimize patient selection schemes for KTN3379 to maximize its clinical benefits. Mol Cancer Ther; 15(4); 689-701. ©2016 AACR.